The invention relates to a method for the fermentative production of L-cysteine and derivatives of said amino acid L-cystine and 2-methylthiazolidine-2,4-dicarboxylic acid.
L-Cystine is a disulfide that is formed on the oxidation of two molecules of L-cysteine. This reaction is reversible, which means that L-cystine can be converted by reduction back into L-cysteine.
2-Methylthiazolidine-2,4-dicarboxylic acid (thiazolidine) is the condensation product of L-cysteine and pyruvate, which is formed in a purely chemical reaction (U.S. Pat. No. 5,972,663A). This reaction is likewise reversible. Thus, the thiazolidine can be decomposed, e.g. in acids at elevated temperature, back to its starting components.
The amino acid L-cysteine is of economic importance. It is used, for example, as food additive (in particular in the baking agent industry), as feedstock in cosmetics, and also as raw material for the production of pharmaceutical active ingredients (in particular N-acetylcysteine and S-carboxymethylcysteine).
L-Cysteine, in all organisms, takes a key position in sulfur metabolism and is used in the synthesis of proteins, glutathione, biotin, lipoic acid, methionine and other sulfur-containing metabolites. In addition, L-cysteine serves as a precursor for the biosynthesis of coenzyme A. The biosynthesis of L-cysteine has been studied in bacteria, in particular in enterobacteria, in detail and is extensively described in Kredich (1996, Biosynthesis of cysteine, p. 514-527. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.).
An important precursor from central metabolism in L-cysteine biosynthesis is 3-phosphoglycerate, an intermediate of glycolysis. L-Cysteine is therefore, together with L-serine and glycine, considered as part of the phosphoglycerate family. The proteinogenic amino acids L-methionine, L-isoleucine and L-threonine, in contrast, belong to the oxaloacetate family, since the shared precursor from central metabolism is the citric acid cycle intermediate oxaloacetate. Proceeding from a carbon source such as, for example, glucose, the material flow proceeds via these two different branches of the assimilatory amino acid metabolism very largely independently of one another. However, certain interactions of intermediates or end products of the phosphoglycerate family with individual biosynthesis steps of the oxaloacetate family are known. For instance, it has been described that homoserine dehydrogenase I, the thrA gene product which catalyzes the reduction of L-aspartate semialdehyde to L-homoserine, can be inhibited not only by L-serine, but also by L-cysteine (Hama et al., 1991, J. Biochem. 109, 604-8; Datta, 1967, Biochemistry 58, 635-41). Furthermore, it is known that the assimilatory threonine deaminase IlvA is inhibited by L-cysteine (Harris, 1981, J. Bacteriol. 145, 1031-35). In addition, L-serine—analogously to L-threonine—can serve as a substrate of the IlvA gene product (Rasko et al., 1971, Eur. J. Biochem. 21, 424-27).
Although it is described in various patents that L-isoleucine can be added to the culture medium in the fermentative production of amino acids of the phosphoglycerate family (EP1233067, EP1382684), L-isoleucine is cited therein only as one of various possible additions. The fact that L-isoleucine could possibly have a beneficial effect on the amino acid production, however, is not indicated or rendered obvious at any point.
In Dassler et al. (2000, Mol. Microbiol. 36: 1101-1112), the addition of the amino acids L-methionine, L-isoleucine and L-leucine to the medium of the preculture (in each case 0.3 g/l) in the fermentative production of amino acids of the phosphoglycerate family is described. However, these amino acids were not added to the medium of the main culture in the fermenter. Here also, there is no indication that the amino acid supplementation of the preculture medium could have a beneficial effect on the production of cysteine or acetylserine.
In addition to the classical production of L-cysteine by means of extraction from keratinaceous material such as hairs, bristles, horns, hooves and feathers, or by means of biotransformation by enzymatic conversion of precursors, some years ago, a method was also developed for the fermentative production of L-cysteine. The prior art with respect to the fermentative production of L-cysteine by microorganisms is described, e.g., in U.S. Pat. Nos. 6,218,168B1, 5,972,663A, US20040038352A1, CA2386539A1, US20090053778A1 and US20090226984A1. Bacterial host organisms used here are, inter alia, strains of the genera Corynebacterium and also representatives of the Enterobacteriaceae family such as, e.g., Escherichia coli or Pantoea ananatis. 
In addition to the classical procedure to arrive at improved L-cysteine producers by mutation and selection, genetic modifications to the strains were also performed in a targeted manner in order to achieve effective L-cysteine overproduction.
Thus, the introduction of a cysE allele, which encodes a serine-O-acetyl transferase having reduced feedback inhibition by L-cysteine, led to an increase in cysteine production (U.S. Pat. No. 6,218,168B1; Nakamori et al., 1998, Appl. Env. Microbiol. 64: 1607-1611). The formation of O-acetyl-L-serine, the direct precursor of L-cysteine, is substantially decoupled from the L-cysteine level of the cell by a feedback-resistant CysE enzyme.
O-Acetyl-L-serine is formed from L-serine and acetyl-CoA. Therefore, providing L-serine in a sufficient amount for L-cysteine production is of great importance. This can be achieved by introducing a serA allele which encodes a 3-phosphoglycerate dehydrogenase having reduced feedback inhibition by L-serine. As a result, the formation of 3-hydroxypyruvate, a precursor of L-serine, is substantially decoupled from the L-serine level of the cell. Examples of such SerA enzymes are described in EP0620853 and US2005009162A2.
In addition, it is known that the L-cysteine yield in the fermentation can be increased by attenuating or destroying genes which encode L-cysteine-degrading enzymes, such as, e.g., the tryptophanase TnaA or the cystathionine-β-lyases MalY or MetC (EP1571223).
Increasing the transport of L-cysteine out of the cell is a further possibility for increasing the product yield in the medium. This can be achieved by overexpression of what are termed efflux genes. These genes encode membrane-bound proteins which mediate the export of L-cysteine out of the cell. Various efflux genes have been described for L-cysteine export (U.S. Pat. No. 5,972,663A, US20040038352A1).
The export of L-cysteine out of the cell into the fermentation medium has many advantages:                1) L-Cysteine is continuously withdrawn from the intracellular reaction equilibrium with the consequence that the level of this amino acid in the cell is kept low, and therefore there is no feedback inhibition of sensitive enzymes due to L-cysteine:                    (1) L-cysteine (intracellular)⇄L-cysteine (medium)                        2) The L-cysteine secreted into the medium is oxidized to the disulfide L-cystine in the presence of oxygen which is introduced into the medium during the culturing (U.S. Pat. No. 5,972,663A):                    (2) 2 L-cysteine+½O2⇄L-cystine+H2O Since the solubility of L-cystine in aqueous solution at a neutral pH is only very low, especially in comparison to L-cysteine, the disulfide precipitates out even at a low concentration, and forms a white precipitate:            (3) L-cystine (dissolved)⇄L-cystine (precipitate) Owing to the precipitation of L-cystine, the level of the product dissolved in the medium is lowered, as a result of which also in each case the reaction equilibrium of (1) and (2) is shifted to the product side.                        3) The technical expenditure for purifying the product is markedly lower when the amino acid can be obtained directly from the fermentation medium, than when the product accumulates intracellularly and a cell digestion must proceed first.        
The expression “total cysteine”, in the context of this invention, combines L-cysteine and the compounds L-cystine and thiazolidine formed therefrom, which are formed during fermentation and accumulate in the culture supernatant and in the precipitate.
In addition to genetic modification of the L-cysteine production strain, optimizing the fermentation method, i.e. the type and manner of culturing the cells, also plays an important role in the development of an efficient production process.
In this case, various culture parameters such as, e.g., the type and dosage of the carbon and energy sources, the temperature, the supply with oxygen (DE102011075656A1), the pH, and also the composition of the culture medium, can affect the product yield and/or the product spectrum in the fermentative production of L-cysteine.
On account of the continually increasing raw material and energy costs, there is constantly the need to increase the product yield in the production of L-cysteine, in order, in this manner, to improve the economic efficiency of the process.